Strong evidence from family and twin studies demonstrates that major depressive disorder (MDD) is heritable, yet there has been limited progress in identifying the actual genes involved. A separate, perhaps overlapping set of genes is expected to play a role in individual variation in treatment response in MDD. We seek to characterize genetically, using a large set of markers in many genes, patients who differ in their response to standard treatments as defined in the STAR*D protocol. Our initial focus was on two sets of genes most likely to play a role in the etiology of major depression: 1) genes selected on neurobiological grounds because of their known involvement in pathways thought to be important in mood disorders;and 2) genes implicated primarily by their positions within genomic regions implicated by genetic linkage or association studies of major mood disorders and related conditions. Our ultimate goal is to study markers representing every common functional variation in the human genome, once such genome-wide studies become technologically feasible. We utilize state-of-the-art, high-throughput genotyping methods as well as sophisticated methods of genetic analysis that take into account haplotypes and multi-locus interactions in addition to standard, single-marker analyses. Primary comparisons are performed with the group of cases who respond to citalopram and those who do not, but analyses are done in other treatment groups as well, depending on sample size. In the first years of this project, we completed genotyping 738 markers in a set of 68 candidate genes selected by an expert panel. Results implicated several of these genes in treatment outcome and other genes that contribute to adverse effects. For example, we identified a marker in the gene encoding the serotonin 2A receptor (HTR2A) that was reproducibly associated with both response and remission during 6-12 weeks of therapy with citalopram. We also identified markers near two genes encoding the ionotropic glutamate receptors, GRIK2 and GRIA3, that seem to be associated with treatment-emergent suicidal ideation. We also discovered additional markers that predict treatment outcome. One marker is in the gene GRIK4, that encodes yet another ionotropic glutamate receptor. A second marker is in the gene FKBP5, which encodes a protein involved in the trafficking of glucocorticoid receptors, key molecules in the stress response system. This latter finding confirms an earlier report implicating this gene in an independent sample of inpatients with major depression. We also discovered genetic markers that help identify those at risk for sexual dysfunction during antidepressant therapy. Sexual dysfunction, such as erectile dysfunction, is one of the most common complaints during treatment with modern selective serotonin reuptake inhibitor antidepressants. We also expanded our study of treatment-emergent suicidal ideation, and identified additional markers that increase risk of this worrisome event. Taken together with our previous findings, these markers can identify a few individuals at substantially increased risk, who may benefit from closer monitoring or alternative treatments. In the past year, we joined a collaboration with investigators from Harvard, the Max Planck Institute Munich, UCSF, and University College London to carryy out a meta-analysis of STAR*D along with the two other genome-wide association studies of antidepressant outcome that have been completed, known as MARS and GENDEP. Despite greater power of this combined sample to confer to uncover association with common genetic markers, no genome-wide significant associations were uncovered. We conclude that no common alleles of large effect on antidepressant outcome exist in these samples. With the support of a Bench to Bedside Award, we have completed genotyping of 1000 participants in the HALT-C trial, who recieved alpha interferon for viral hepatitis. We are seeking to find genetic markers of mood and other psychiatric symptoms that commonly emerge during alpha interferon therapy. Initial results indicate that no single gene predicts depressive symptoms, but indivduals who develop depression during interferon therapy carry a higher burden of the same risk alleles seen in people who develop major depression without interferon exposure. This suggests the genes contribute to a general vulnerability to depression that may emerge after exposure to any of many different provocatve factors. In the coming year, we will take advantage of new, high-throughput sequencing methods to test for rarer alleles that may exert larger effects, at least on treatment resistance. It is possible that the focus on symptom improvement and common alleles has led the field astray. Patients who respond to antidepressant treatment constitute a mixture of true responders, placebo responders, and spontaneous remitters. Patients who fail to respond to multiple treatments may be less heterogeneous, since placebo responders and spontaneous remitters are removed. Truly treatment-resistant depression (TRD) still needs to be differentiated from non-adherence, intolerance, delayed response, and comorbidities that interfere with treatment, but this distinction can generally be accomplished in prospective studies like STAR*D. Over 4,000 patients enrolled in STAR*D and of these, only 10% were deemed treatment-resistant, and only 3% were deemed highly treatment-resistant. Rarer alleles may show larger effects, especially among patients with unusual treatment outcomes. Sequencing studies may uncover alleles that play a major role in a minority of patients. Few patients will carry such alleles, but the genes involved will point to attractive new drug targets. Although family designs are impractical for pharmacogenetic studies, unrelated cases who sit at the extremes of the response distribution may be particularly informative. This rare outcomes/rare alleles strategy has already been successful in other fields, but has not yet been tried in antidepressant outcome studies. With the support of a K99 award to Gonzalo Laje, we are also commencing a combined MRS imaging and pharmacogenetics study of 100 patients undergoing outpatient treatment for major depressive disorder. The goal is to determine wheher there is an MRS signature of treatment response and the degree to which this is under genetic influence.